The Raf family of serine/threonine protein kinases become hyperphosphorylated upon treatment of appropriate cell lines with various agents, including growth or differentiation factors. This phosphorylation is concurrent with enhancement in kinase activity. In order to better understand this mechanism of activation, the major phosphorylation sites were mapped for Raf-1, and the major in vitro autophosphorylation site was mapped for B-Raf. Since one of the agents activating Raf-1 kinase was the protein kinase C (PKC)-alpha activator 12-0-tetradecanoylphorbol-13-acetate, the role of PKC in Raf-1 activation was investigated. It was found that PKC-alpha is involved directly in activation of the Raf-1 kinase through phosphorylation of two serine residues. As for other Raf kinase kinases, there is also a potential cdc2 target site in B-Raf that we are in the process of investigating. In an effort to investigate the role of activated Raf kinases also in downstream events, we have started characterization of the phosphorylation sites of mitogen-activated protein kinase kinase, a recently identified substrate.